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We have employed a direct cDNA selection methodology to isolate transcribed sequences encoded in the human chromosomal interval Xq26 that contains the gene for X-chromosome linked albinism deafness syndrome (ADFN). ADFN had been previously mapped to an 8 centi Morgan region on chromosome Xq26. We have constructed six cDNA libraries specific to six YACs mapping to a 1.5 mb span at the distal boundary of the ADFN locus. The YAC specific libraries were characterized for the presence of unique cDNAs. We have identified 15 transcribed sequences from the selected cDNA libraries. These cDNAs matched to three well characterized sequences corresponding to steroid 5-alpha reductase, ribosomal protein L28, and a short transcript that has been shown to be expressed in human brain cortex. Seven of the cDNAs matched to expressed sequence tags or other sequences of unknown function, and five cDNAs shared no homology with sequences in the public data bases. Each one of these sequences was represented as 3-10 clones in the set that was subjected to sequencing. Further characterization of these transcribed sequences may indicate potential candidates responsible for ADFN. We have discussed the utility of cDNA selection methodology in assembling transcript maps and identifying potential candidates for genetic deafness.

We isolated cDNA fragments encoded in an X-chromosome specific YAC from the locus DXS995 by using direct cDNA selection method. Several of the selected cDNA fragments were identical to various exons of cyclophilin A gene. Hybridization of the selected cyclophilin cDNA fragments to the target YAC, presence of these sequences in an X-chromosome specific phage library and absence of a cross hybridizing fragment in a cell line (XL-45) that contains deletions of the interval Xq21, demonstrates that a cyclophilin like sequence is present in the human X-chromosome.

The 3prime terminal 1255nt sequence of Physalis mottle virus (PhMV) genomic RNA has been determined from a set of overlapping cDNA clones. The open reading frame (ORF) at the 3prime terminus corresponds to the amino acid sequence of the coat protein (CP) determined earlier except for the absence of the dipeptide, Lys-Leu, at position 110-111. In addition, the sequence upstream of the CP gene contains the message coding for 178 amino acid residues of the C-terminus of the putative replicase protein (RP). The sequence downstream of the CP gene contains an untranslated region whose terminal 80 nucleotides can be folded into a characteristic tRNA-like structure. A phylogenetic tree constructed after aligning separately the sequence of the CP, the replicase protein (RP) and the tRNA-like structure determined in this study with the corresponding sequences of other tymoviruses shows that PhMV wrongly named belladonna mottle virus [BDMV(I)] is a separate tymovirus and not another strain of BDMV(E) as originally envisaged. The phylogenetic tree in all the three cases is identical showing that any subset of genomic sequence of sufficient length can be used for establishing evolutionary relationships among tymoviruses.

Purpose: To determine if there is a racial difference in prostate cancer related to loss of heterozygosity (LOH) on chromosome 8p12–23, the region most frequently altered in prostate cancer. Methods and Materials: A total of 51 prostate cancer patients, consisting of 23 African Americans and 28 Caucasians, were included in this study. All patients underwent radical prostatectomy, and patients in the two racial subgroups were matched for median serum PSA, Gleason score, and pathological stage of cancer. Paired normal prostate and cancer tissue DNA was isolated and amplified with 13 polymorphic markers mapped to 8p12–23 by radiolabeled polymerase chain reaction. The amplified products were resolved by polyacrylamide gel electrophoresis, autoradiographed, and analyzed for allelic losses. Results: The overall incidence of LOH at 8p12–23 was 53%, and 16% showed homozygous deletions. The incidence of LOH in Caucasians was 68% compared to 35% in African Americans. On univariate ( p = 0.02) and multivariate logistic regression analysis ( p = 0.02), only Caucasian race was a significant predictor for LOH. The other clinicopathologic parameters did not have any significant effect on incidence of LOH. Conclusion: These results highlight the independent influence of Caucasian race on incidence of LOH at 8p12–23, and suggest that genetic differences at specific tumor suppressor loci may be a factor responsible for racial variations observed in prostate cancer.

We have isolated and characterized the cDNA for eZNF, a zinc finger gene expressed in human inner ear, from a kinetically enriched human inner ear cDNA library. The sequence of full length cDNA was determined and its expression pattern characterized. A high degree of homology is shared between eZNF and rat transcription factor Kid-1. It belongs to the C2H2 class of zinc finger genes, contains a Kruppel-associated box (KRAB) domain near the N-terminus, and has consensus sites for phosphorylation. The gene is expressed in kidney and inner ear structures of mouse and human as determined by Northern blot analysis. In situ hybridization was used to demonstrate specific expression of the mouse eZNF homologue in epithelial layers of the saccule, semicircular canals, and the cochlea of newborn mice. The genomic clone corresponding to the cDNA was isolated and used for fluorescence in situ hybridization to localize it to human chromosome 5qter. The identification of genes expressed in human inner ear by representational difference analysis, their chromosomal location, and expression pattern of their homologues in developing mouse inner ear comprise a strategy that can potentially identify genes important in hearing and deafness.

The genome of physalis mottle tymovirus (PhMV) is 6673 nucleotides long and is rich in cytosine residues (40.58%) like other tymoviruses. The organization of the genes is also similar to that of five other tymoviruses whose sequences are known. However, PhMV has the longest 3' noncoding region as well as the longest replicase (RP) ORF. The RP sequences are similar to those of other tymoviruses (48-60% identity) whereas the coat proteins (CP) and the overlapping proteins (OP) are conserved to a lesser extent (30-50% and 26-34% respectively). A tetra peptide "GILG" was found to be present in all the tymoviral OPs. The PhMV RP also possesses the methyl transferase, polymerase and the helicase motifs found in all the Sindbis-like super group of plant viruses. A phylogenetic analysis of the six tymoviral sequences revealed that they do not have a rigid hierarchical similarity relationship.

We have used oligonucleotides corresponding to conserved regions of protein kinase regulators of 14-3-3 gene family as primers to amplify these genes from cDNAs constructed from the human fetal inner ear. Sequence characterization of clones revealed that the 14-3-3 cDNA library from the fetal inner ear has high abundance of clones encoding a protein kinase regulator (theta subtype), a member of 14-3-3 gene family, and relatively lower abundance of clones for two other members of the gene family. One of these genes is identical to the eta subtype of human 14-3-3 genes; there is no cloned gene for the other subtype in the human 14-3-3 gene family in the nucleic acid data bases. A sequence homology search revealed that the latter shared significant homology with the gamma subtype of the rat 14-3-3 family. On the basis of the sequence data, it appears that this clone represents a human homolog of the rat gamma subtype. The results demonstrate the expression of 14-3-3 genes in the inner ear, characterize a human homolog of the rat gamma subtype of 14-3-3, implicate these proteins in ear development, and indicate the utility of gene family polymerase chain reaction (PCR) for investigating gene expression in specific tissues.

We have used representational difference analysis (RDA) for subtractive hybridization of oligo dT primed directionally cloned cDNA libraries from human inner ear tissue and a B-lymphoblast cell line. Two rounds of subtraction-amplification, followed by differential hybridization of selected clones led to the isolation of genes which were specific to the ear. Sequence analysis of randomly chosen clones revealed the presence of a histidine rich Ca2+ binding protein, human dynamin, collagen type 1A1, collagen type 2A1, SPARC, human growth hormone, and several specific genes which had no sequence homology in the data base. Furthermore, to apply these techniques for isolating genes specific to distinct inner ear structures and/or cell types of inner ear for which the starting tissue material is limiting, we have used a modified PCR based protocol to construct representative cDNA libraries. We have characterized a cDNA library constructed from small amounts of inner ear tissues recovered by ablative surgical procedure involving labyrinthectomy. The potential application of these protocols for isolating genes involved in hearing and deafness is discussed.

We have used a direct cDNA selection protocol to isolate expressed sequences from yeast artificial chromosome clones that contain approximately 900 Kb of genomic DNA from Xq21 band that is deleted in contiguous gene syndromes comprising of mixed deafness associated with stapes fixation (DFN3). In addition to identifying Brn4 (POU3f4), a POU domain containing transcription factor that is involved in DFN3 phenotype, we have isolated seven short fragment cDNAs mapping to the deleted region. Some of the selected fragments showed X-chromosome specificity and hybridized to autosomal DNA fragments, indicating the presence of a low abundance interspersed repeat in the cDNAs or their homology to some uncharacterized family of genes. In conformity with the inertness of Xq21 band our results demonstrate that the region encodes far less than the average density of genes in other parts of the genome.

Physalis mottle tymovirus (previously named belladonna mottle virus, Iowa strain) RNA was cross-linked to its coat protein by exposure of the intact virus to ultraviolet light. The site of cross-linking of the coat protein with the RNA was identified as Lys-10 by sequencing the oligonucleotide-linked tryptic peptide obtained upon HPLC separation subsequent to enzymatic digestion of the cross-linked and dissociated virus. Three monoclonal antibodies PA3B2, PB5G9, and PF12C9, obtained using denatured coat protein as antigen, cross-reacted effectively with the intact virus indicating that the epitopes recognized by these monoclonals are on the surface of the virus. Using the peptides generated by digestion with CNBr, clostripain, V-8 protease, or trypsin and a recombinant protein lacking the N-terminal 21 residues expressed from a cDNA clone, it was shown that PA3B2 recognizes the sequence 22-36 on the coat protein while PB5G9 and PF12C9 recognize region 75-110. These results suggest that Lys-10 is one of the specific sites through which the RNA interacts in the intact virus. The polypeptide segment (region 22-36) following this buried portion as well as the epitope within the region 75-110 are exposed in the intact virus. These observations are consistent with the canonical beta-barrel structure observed in certain other plant viruses.